5-methyl-3-(p-guanidinophenyl)-4-isoxazolylcarboxylic acid



United States Patent 3,257,411 -METHYL-3-(P-GUANIDlNOPHENYL)-4- ISOXAZULYLCARBOXYLIC ACID Arthur A. Patchett, Metuchen, and Edward F. Rogers,

Middletown, NJ., and William J. Leanza, Staten Island, N.Y., assignors to Merck & 'Co., Inc., Rahway,

N.J., a corporation of New Jersey No Drawing. Original application June 14, 1963, Ser.

No. 287,783. Divided and this application May 14, 1965, Ser. No. 464,891

1 Claim. (Cl. 260-307) wherein R represents an organic radical, have been found to be invaluable in the therapy of infections, particularly those due to gram-positive bacteria. Thus, penicillins such as benzylpenicillin and phenoxyrnethylpenicillin are widely used in the treatment of various infections. Although penicillins have found widespread use, the presently-known penicillins do sufier from one or more disadvantages. For example, some of the penicillins, such as benzylpenicillin, have comparatively low in vivo activity even when administered parenterally. Some of the penicillins are ineffective against certain gram-negative strains of bacteria and the use of these penicillins must therefore be limited to the treatment of infections caused by bacteria susceptible to the action of the penicillin. Accordingly, the search has continued to find other penicillins which do not possess the disadvantages of the known penicillins.

It is an object of this invention to provide penicillins and their salts which are particularly effective against gram-negative bacilli.

Another object is to provide penicillins which have high in vivo activity against infections by such microorganisms.

Still another object is to provide new intermediates and processes for the preparation of such penicillins.

Other objects will be apparent from the detailed description of this invention hereinafter provided.

In accordance with this invention, it is now found that penicillins wherein the acyl group contains a guanidinoaryl or a guanidinomethylaryl radical, and the non-toxic acid salts thereof are valuable anti-bacterial products. These products, which may also be termed 6-(guanidinoarylacylamino)-penicillanic acids, in addition to having pronounced antibacterial activity against gram-negative bacilli, possess high in vivo activity when compared with the corresponding penicillins lacking a guanidino substituent. These new penicillins are of the formula:

COOH

wherein R is an organic radical containing a guanidinoaryl or a guanidinomethylaryl substituent.

The new penicillins of the present invention are prepared by reacting a suitable ester of 6-aminopenicillanic acid'with a novel organic carboxylic acid having a guanidinoarylor a guanidinomethylaryl substituent to produce the corresponding ester of'the G-(guanidinoarylacylamino)-penicillanicacid and then removing the pro tective ester by catalytic hydrogenation to obtain the desired free acid.

Esters of 6-aminopenicillanic acid suitable for use in carrying out the processes of this invention are those which can be readily cleaved by catalytic hydrogenation.

Suitable esters of 6-aminopenicillanic acid that might be mentioned include allyl, benzyl, substituted benzyl, phenyl, substituted phenyl and the like. Since the benzyl ester is most conveniently prepared it is presently preferred to use this ester in the preparation of the new penicillins of this invention.

Alternatively, the new penicillins of the present invention are prepared by reacting 6-aminopenicillanic acid or a salt thereof with an acid halide of an organic acid having a guanidinoarylor a guanidinomethylaryl substituent to produce the 6-(guanidinoarylacylamino)-penicillanic acid or its alkali metal salt directly.

The novel guanidinoaryl or guanidinomethylaryl-substituted organic acids or acid halides used as the acylating agent in the preparation of the new penicillins have the formula:

wherein R is a guanidinoaryl, a guanidinomethylaryl, a guanidinoaralkyl or a guanidinomethylaralkyl substituent and X is a hydroxy or a halo radical.

In preparing the new guanidino-substituted carboxylic acids or acid halides used. as the acylating agents, the appropriate amino acid is converted to the corresponding carboxylic acid having a guanidino substituent in place of the amino substituent, and the resulting guanidino-substituted carboxylic acid recovered from the reaction mixture. The corresponding acid halide is prepared by reaction of the thus-obtained guanidinocarboxylic acid with a halogenating agent, e.g., thionyl chloride, in the presence of a tertiary amine.

The new guanidino-substituted carboxylic acids or acid halides employed as intermediates in the preparation of the novel guanidino-substituted penicillins by reaction with 6-aminopenicillanic acid or esters thereof, are of four general types, i.e., a guanidinoarylcarboxylic acid, a guanidinomethylarylcarboxylic acid, a guanidinoaralkylcarboxylic acid or a guanidinomethylaralkylcarboxylic acid.

In accordance with our invention, the guanidino-substituted carboxylic acid may be prepared from the corresponding amino-substituted carboxylic acid by contacting the selected amino acid with s-methylisothionitrourea to yield as an intermediate product the corresponding nitroguanidinocarboxylic acid, which is then converted by catalytic hydrogenation to the desired guanidino-substituted carboxylic acid. The guanidino-substituted carboxylic acid prepared in this manner may be converted to the corresponding acid chloride, e.g., by reaction with thionyl chloride.

In accordance with a preferred method of preparing guanidinoarylcarboxylic acids and guanidinoaralkylcarboxylic acids, the selected aminoarylor aminoaralkylcarboxylic acid is intimately contacted with benzoyl cyanamide and the resulting product hydrolyzed to the desired guanidinoarylor guanidinoaralkylcarboxylic acid. Thus, for example, p-guanidinobenzoic acid is prepared by intimately contacting p-aminobenzoic acid and benzoyl cyanamide followed by hydrolysis of the reaction product, 5- methyl 3 (p g'uanidinophenyl) 4 isoxazolyl carboxylic acid is prepared by the reaction of 5-rnethyl-3-(pea aminophenyl)-4-isoxazolylcarboxylic acid, and benzoyl cyanamide followed by hydrolysis of the reaction product to yield the desired guanidino-substituted acid, and p-guanidinophenylacetic acid is prepared by contacting p-aminophenylacetic acid with benzoyl cyanamide followed by hydrolysis of the resulting reaction product to the desired guanidino acid.

In a preferred method of preparing the guanidinomethylarylcarboxylic acids and the guanidinomethylaralkylcarboxylic acids, the selected aminomethylarylcarboxylic acid or the aminomethylaralkylcarboxylic acid is reacted with o-methylisourea in alkaline solution to produce the corresponding guanidinomethylarylcarboxylic acid or guanidinomethylaralkylcarboxylic acid. Thus, for example, p-guanidinomethylbenzoic acid is prepared by reaction of p-aminomethylbenzoic acid and o-methylisourea in alkaline solution, and p-guanidinomethylphenylacetic acid is similarly obtained from p-aminomethylphenyl acetic acid.

In accordance with the above procedures, the following novel guanidino-substituted carboxylic acids, the corresponding acid halides, and salts thereof, are readily prepared from the corresponding amino acids:

2-phenyl-4-guanidinobenzoic acid; 4-guanidino-2-ethoxy-l-naphthoic acid; 2,6-dirnethoxy-4-guanidino-benzoic acid; 2phenyl-4-guanidinomethylbenzoic .acid; 4-guanidinomethyl-2-ethoxy-l naphthoic acid; 2,6 dimethoxy-4-guanidinomethylbenzoic acid; 4-guanidinophenylacetic acid; 4-guanidinophenoxyacetic acid; 2-(4-guanidinophenoxy) -propionic acid; 5-methyl-3- (4-guanidinophenyl) -4-isoxazolylcarboxylic acid; p-guanidinophenyl-aeaminoacetic acid; 2-(4-guanidinophenoxy) -butyric acid; 5-methyl-3- 2-chloro-4-guanidinophenyl -4-isoxazolylcarboxylic acid; 4-guanidinomethylphenylacetic acid; 4-guanidinomethylphenoxyacetic acid; 2- (4-guanidinomethylphenoxy -propionic acid; 5-methyl-3- (4-guanidinomethylphenyl) -4-isoxazolylcarboxylic acid; p-guanidinomethylphenyl-a-aminoacetic acid; 2-(4-guanidinomethylphenoxy)-butyric acid; 5-methyl-3- 2-chloro-4-guanidinomethylphenyl -4- isoxazolylcarboxylic acid.

By reaction of an acid selected from the foregoing list with a halogenating agent, e.g., thionyl chloride, there is formed the corresponding acid halide or salt thereof, e.g., 2-phenyl-4-guanidinobenzoyl chloride hydrochloride.

The reaction of the 6-aminopenicillanic acid ester and the guanidino-substituted organic acid is conveniently carried out by contacting the reactants in the presence of a carbodiimide such as 1,3-dicyclohexyl carbodiimide or 1,3-diisopropyl carbodiirnide, in the presence of a suitable solvent at room temperature. After completion of the reaction, the urea formed from the dicarbodiimide is separated from the reaction mixture and the desired penicillin ester is recovered from the resulting solution in accordance with methods well known in this art.

The ester of the new penicillin so obtained is readily converted to the free acid by catalytic hydrogenation. This catalytic hydrogenation is preferably carried out using a noble metal catalyst such as palladium, platinum or rhodium; preferably supported on an inert carrier such as carbon, barium carbonate, diatomaceous earth, and the like. After completion of the hydrogenation, the free penicillanic acid is recovered from the hydrogenation reaction mixture by procedures known in the art.

The new compounds of our invention include penicillins which contain a guanidino-substituted aryl substituent attached to the acyl portion of the penicillin molecule. These new penicillins may be prepared as described above by reaction of an appropriately substituted alkanoic acid or acid halide with either a 6-aminopenicillanic acid or an ester thereof. The organic acid or acid halide selected is preferably one having a monoeyclic aryl substituent at tached to the carboxylic acid wherein the monocyclic aryl substituent is further substituted by a guanidinoor a guanidinomethyl radical. In addition to theguanidinoor guanidinomethyl substituent, the carboxylic acid or acid halide may contain other substituents attached to the monocyclic aryl nucleus or to some other position of the alkanoic acid carbon chain. Examples of guanidinoarylor guanidinomethylaryl penicillins as well as the guanidinoaralkylor guanidinomethylaralykyl penicillins also include those in which a hetero atom or a heterocyclic ring is interposed between the aryl nucleus and the amide linkage of the penicillin molecule. Specific penicillins that are included in the scope of our invention are:

2-phenyl-4-guanidinophenyl penicillin; 2-ethoxy-4-guanidinonaphthyl penicillin; 2,6-dimethoxy-4-guanidinophenyl pencillin; 2-phenyl-4-guanidinomethylphenyl penicillin; 2-ethoxy-4-guanidinomethylnaphthyl penicillin; 2,6-dimethoxy-4-guanidinomethylphenyl penicillin; 4-guanidinobenzyl penicillin; 4-guanidinophenoxymethyl penicillin; ot-(4-guanidinophenoxy)-ethyl penicillin; 5 -methyl-3- (4- guanidinophenyl) -4-isoxazolyl penicillin; a-amino-p-guanidinobenzyl penicillin; 06-- (4-guanidinophenoxy -propyl penicillin; 5-methyl-3-(2-chloro-4-guanidinophenyl)-4-isoxazolyl penicillin; 4-guanidinomethylbenzyl penicillin; 4-guanidinomethylphen oxymethyl penicillin; a- (4-guanidinomethylphenoxy -ethyl penicillin; 5 -methyl-3- (4-guanidinomethylphenyl) -4-isoxazolyl penicillin; a-amino-p-guanidinomethylbenzyl penicillin; a-(4-guanidinomethylphenoxy) -propyl penicillin; 5-methyl-3 Z-chloro-4-guanidinomethylphenyl -4- isoxazolyl penicillin.

These penicillins can be obtained in the form of their inner salts, or they can be reacted with acids to obtain the corresponding penicillin acid salts. The inner salts and the non-toxic acid salts can be admixed with suitable non-toxic liquid or solid pharmaceutical carriers for administration to man and animals. These pharmaceutical preparations can contain other active therapeutic agents, and may be administered either orally or parenterally.

The new penicillins and their non-toxic salts are valuable antibacterial agents which are useful therapeutic agents in poultry and other animals, including man, in the treatment especially of infections caused by susceptible gram-positive and gram-negative bacteria. They are also useful as nutritional supplements in animal feeds.

The following examples are provided as illustrations of the methods of preparing the novel penicillins and the non-toxic salts thereof as well as the new guanidino-substituted organic acid intermediates in the production of the new penicillins of our invention.

EXAMPLE 1 p-Guanidinophenyl penicillin Approximately 650 mg. of solid p-guan'idinobenzoyl chloride hydrochloride isadded in small amounts to a stirred solution of 648 mg. of 6-aminopenicillanic acid in 8.5% aqueous sodium bicarbonate solution. The pH of the reaction mixture is maintained in the range of 7.8 during the course of the reaction by the continuous addition of a saturated aqueous solution of sodium bicarbonate. The reaction mixture is then allowed to stir for approximately 45 minutes and the formed p-guanidinophenyl penicillin precipitates from the reaction mixture. The precipitated pro-duct is recovered by filtration and purified by recrystallization from an acetone-water mixture. The product thus obtained inhibits Staphylococcus aureus at a concentration of 3.9 mg./rnl.

The p-guanidinobenzoyl chloride hydrochloride used as the starting material is prepared from p-aminobenzoic acid in the following manner. A solution of 1.5 g. of p-aminobenzoic acid, 1.62 g. of benzoyl cyanamide in 5 ml. of

ethanol are evaporated to dryness at steam bath tempera' An additional 5 ml. of ethanol isadded and the The residue ture. evaporation repeated in a similar manner. is stirred for 1 hour in 5% aqueous sodium bicarbonate solution, and filtered. To the filtrate is added 4 equivalents of sodium hydroxide, and the resulting vsolution maintained at reflux temperature for approximately 20 minutes. The solution is then decolorized with activated charcoal and to the decolorized filtrate is added an addi- 5-methyl-3- (p-guaniziinophenyl) -4 -isxaz0lyl penicillin A mixture of 400 mg. of -methyl-3-(p-guanidinophenyl)4-isoxazolylcarboxylic acid and .4 ml. thionyl chloride are stirred at reflux temperature for approximately l5 minutes. The reaction mixture is concentrated in vacuo to remove excess thionyl chloride, leaving as a residue 5-methyl-3-(p-guanidinophenyl) 4 isoxazolylcarboxylic acid chloride hydrochloride. The residual acid chloride is dissolved in 5 ml. of dry acetone and the resulting solution added to a solution containing 283 mg. of 6-aminopenicillanic acid dissolved in a small amount of saturated sodium bicarbonate solution. The mixture is stirred for approximately 30 minutes at a temperature of 0 to 5 while adding aqueous sodium bicarbonate solution to maintain the pH between about 7 and 8, and the precipitate which forms is removed by filtration and discarded leaving the product dissolved in the filtrate. The filtrate is concentrated under vacuum untilthe 5-methyl-3-(pguanidinophenyl)4-isoxazolyl penicillin precipitates and is recovered by filtration. This product inhibits Staphylococcus aureous at a concentration of 3.9 mg./ml.

The 5-methyl-3-(p-guanidinophenyl)4-isoxazolyl carboxylic acid is prepared 'by reaction of p-nitrobenzaldehyde with hydroxyl amine hydrochloride to obtain pnitrobenzaldoxime, which is converted through the pnitrobenzohydroximic chloride by treatment with HCl and chlorine gas and subsequently converted by treatment with ethylacetoacetate in'the presence of a strong base to obtain ethyl [5-imethyl-3-(p-nitrophenyl)-4-isoxazolyl]- carboxylate which is hydrolyzed to the corresponding free acid with sodium hydroxide. The acid obtained is hydrogenated in the presence of a catalyst to produce S-methyl- B-(p-aminophenyl)4-isoxazolylcarboxylic acid which is reacted with benzoyl cyanamide in the presence of sodium hydroxide to yield the desired acid. These reactions are described in detail as follows.

To a stirred solution of 50 g. p-nitrobenzaldehyde in 500 ml. pyridine is added 25.3 g. hydroxylamine hydrochloride. The solution which forms is heated for approximately 1 hour at steam bath temperature. To the solution is added 3 volumes of water and the solution cooled until the product, p-nitr-obenzaldoxime, crystallizes from solution. The crystalline product is recovered by filtration and recrystallized from an ethanol-water mixture.

Approximately 52 g. p-nitrobenzaldoxirne is suspended in 202 ml. of 8.3 N aqueous hydrochloric acid and the resulting mixture cooled to 0 C. Chlorine gas is bubbled through the solution for approximately 1 hour, whereupon crystalline p-nitrobenzaldoxime chloride crystallizes from solution and is recovered by filtration.

To a solution of 18.2 g. sodium methoxide in 113 ml. of methanol is added rapidly 44 g. ethylacetoacetate. After stirring the solution for approximately 10 minutes, it is cooled to 25 C. r

A solution of 51 g. p-nitrobenzaldoxime chloride in 113 ml. methanol is cooled to approximately 10 C. and added to the previously-prepared solution of ethylacetoacetate at a rate such that the temperature of the reaction mixture is held below 0 C. with cooling. The resulting mixture is stirred for approximately 18 hours at 25 C., whereupon an orarige precipitate of ethyl [5-methyl-3-(p nitrophenyl)4-isoxazolyl]carboxylate precipitates and is recovered by filtration. The recovered product is washed with methanol and water and purified by recrystallization from a methanol-ether mixture.

A solution of 43 g. of ethyl [5-methyl-3-(p-nitrophenyl)-4-isoxazoly]carboxylate in 755 ml. of methanol is heated to reflux temperature under an atmosphere of nitrogen, and to it is added a solution of 167 ml., 1 N aqueous sodium hydroxide, and the temperature maintained at reflux for approximately 1 /2 hours. The solution is then cooled to 25 C., 20 ml. of concentrated glacial acetic acid is added, and the solution is concentrated in vacuo until the product precipitates. Water is added to the reaction mixture to complete precipitation of the product which is recovered from solution by filtration, washed and dried. Recrystallization from ethanol gives substantially pure 5-rnethyl-3(p-nitrophenyl)4- isoxazolyl carboxylic acid.

To 1 g. of 5% rhodium-on-carbon catalyst, which is pre-reduced with hydrogen, is added a solution of 2.4 g. 5 -methyl-3-(p-nitrophenyl)4-isoxazolyl carboxylic acid in 250 ml. methanol. The resulting mixture is agitated continuously and hydrogenated under 40 pounds hydrogen pressure until hydrogenation appears to be substantially complete. The catalyst is removed from the solution by filtration and the solvent removed by concentration in vacuo, leaving the product as a residue. The residue is dissolved in ether and triturated with methanol. Further purification by extraction with ethyl acetate and removal of the solvent yields substantially pure S-methyl- 3-(p-aminophenyl)-4-isoxazolylcarboxylic acid.

Approximately 973 mg. of 5-methyl-3-(p-aminophenyl)4-isoxazolylcarboxylic acid and 362 mg. of benzoyl cyanamide are mixed in 5 ml. of ethanol and the solution evaporated to dryness at steamxbath temperature. Addition of 5 ml. of ethanol to the residue and evaporation to dryness are repeated twice. The residue from the reaction mixture is suspended in 5% aqueous sodium bicarbonate solution, stirred for approximately 1 hour and filtered'to remove unreacted starting material. To the filtrate is added approximately 250 mg. of sodium hydroxide and the resulting solution heated at reflux temperature in an atmosphere of nitrogen for approximately 15 minutes. The resulting solution is decolorized with activated charcoal and subsequently treated with approximately 750 mg. of sodium hydroxide and stirred at 25 C. for approximately 30 minutes. The product, 5 methyl-3-(p-guanidinophenyl)4-isoxazolylcarboxylic acid is precipitated by neutralization of the alkaline solution with glacial acetic acid. The precipitated product is recovered by filtration and recrystallized from hot water.

EXAMPLE 3 p-Guanidino'phenoxymethyl penicillin A mixture of 0.208 g. of 6-(p-guanidinophenoxyacetamido)-penicillanic acid benzyl ester hydrochloride and 0.2 g. of palladium-on-carbon catalyst in 10 ml. of 50% aqueous methanol is maintained under 40 pounds per square inch of hydrogen pressure for approximately 2 hours. The reduced reaction mixture is filtered to remove the catalyst and the catalyst is washed with 50% aqueous methanol to remove occluded product. The filtrate and washings containing the desired product are concentrated in vacuo to leave as a residue the desired product. This residue is further purified by countercurrent distribution between N-butanol and water and the substantially pure product, p-guanidinophenoxymethylpenicillin, recovered from the aqueous phase.

The 6 (p guanidinophenoxyacetamido) penicillanic acid benzyl ester hydrochloride is prepared by reaction of p-aminophenoxyacetic acid and s-methylisothionitrourea to yield as an intermediate product p-nitroguanidinophenoxyacetic acid, which is hydrogenated to give as a second intermediate product p-guanidinophenoxyacetic acid hydrochloride, which material is reacted with 6- aminopenicillanic acid benzyl ester in the presence of N,N'-dicyclohexylcarbodiimide in accordance with the following procedure.

A mixture of 1.4 g. of p-aminophenoxyacetic acid and an aqueous solution of sodium hydroxide containing approximately 10 milliequivalents of sodium hydroxide, 2 m1. dimethyl formamide and 1.4 g. of s-methylisothio nitrourea are stirred at 60 C. for 1.5 hours. The resultting solution is cooled and 10 milliequivalents of dilute hydrochloric acid is added to precipitate the formed pnitroguanidinophenoxyacetic acid. The product is recovered by filtration, air dried and dissolved in aqueous sodium hydroxide. The sodium hydroxide solution is filtered to remove extraneous material and washed with ethylacetate. Substantially pure p-nitroguanidinophenoxyacetic acid is crystallized from the aqueous solution by acidification and cooling. The crystalline material is recovered by filtration and dried. M.P. 239 C. (d.).

Approximately 7.3 g. of p-nitroguanidinophenoxyacetic acid is dissolved in 450 ml. of 90% aqueous methanol and reduced with hydrogen at 40 pounds per square inch hydrogen pressure in the presence of 4.5 g. of 10% palladium-on-carbon catalyst until approximately 4 moles of hydrogen are taken up. The catalyst is removed from the reaction mixture by filtration and washed with methanol. The solid catalyst mixture is extracted with boiling water to dissolve the reduced product and filtered. The filtrate containing the product is concentrated to a small volume and treated with dilute hydrochloric acid to produce p-guanidinophenoxyacetic acid hydrochloride, which is recovered by evaporating the aqueous solution to dryness in vacuo. M.P. 170 C.

Approximately 518 mg. of N,N'-dicyc1ohexyl carbodiimide and 779 mg. of 6-aminopenicillanic acid benzyl ester are dissolved in 0.87 ml. of dimethyl formamide. To the resulting solution is added a solution of 500 mg. of

' p-guanidinophenoxyacetic acid hydrochloride and 0.87 ml.

of dimethyl formamide, and the resulting mixture allowed to stand for 1 hour at 25 C. The reaction mixture containing the product is centrifuged to separate the layers and the supernatant layer removed. The residual material is washed with an equal mixture of dimethyl formamide and methylene chloride and again centrifuged and the supernatant layer removed. The supernatant layers are combined and diluted with ether to precipitate an oil containing the desired product. The product is obtained in 'solid form by trituration with ether. The resulting crude solid is dissolved in methylene chloride and reprecipitated with ether to remove unreacted 6-aminopenicillanic acid benzyl ester. The precipitated product is submitted to countercurrent distribution between N-butanol and water, whereby substantially pure G-(p-guanidinophenoxyacetamido)-penicillanic acid benzyl ester hydrochloride is obtained in the butanol phase.

8 EXAMPLE 4 p-Guanidinomethylphenyl penicillin A mixture of 153 mg. of benzyl fi-(p-guanidinomethylbenzamido)-penicillanate hydrochloride, 10 mg. of sodium bicarbonate and 300 mg. of 10% palladium-on-carbon catalyst in 17 ml. of 90% aqueous methanol are hydrogenated at 25 C. under hydrogen at atmospheric pressure for a period of about 45 minutes. The catalyst is removed from the reaction mixture by filtration and washed with methanol to remove occluded product. The combined filtrate and washings are concentrated under reduced pressure to remove the methanol and the residual material containing the product p-guanidinomethylphenyl penicillin is lyophilized to yield amorphous solid product. The product thus obtained inhibits Staphylococcus aureus at a concentration of 7.8 mcg./ml.

The benzyl 6- (p guanidinomethylbenzamido)penicil- 'lanate hydrochloride is prepared by hydrogenation of p-cyanobenzoic acid to obtain as an intermediate product p-aminomethylbenzoic acid which is reacted with o-methyl-isourea in alkaline solution to produce p-guanidinomethylbenzoic acid as a second intermediate product which is, in turn, reacted with benzyl 6-aminopenicillanate in the presence of dicyclohexylcarbodiimide in the following manner.

A solution of 2 g. p-cyanobenzoic acid in 40 ml. of ethanol saturated with ammonia, and 2 g. Raney nickel catalyst are hydrogenated at C. under 1500 pounds per square inch of hydrogen pressure. The hydrogenation reaction mixture is filtered to remove the catalyst and the product recovered from the filtrate by acidification with 2.5 N HCl, whereupon crystalline p-aminomethylbenzoic acid precipitates from solution and is recovered by filtration.

A solution of 1.2 g. of p-aminomethylbenzoic acid in 16 ml. of Water containing 3.76 ml. of concentrated ammonium hydroxide is prepared and mixed with 2.26 g. of o-methylisourea hydrochloride. The resulting reaction mixture is stir-red for about 18 hours and filtered to removed the precipitiated product. The product is dis solved in hot aqueous ethanol and re'precipitated by acidification of the solution to pH 2. The resulting product, substantially pure p-guanidinornethylbenzoic acid bydrochloride, is recovered by filtration and dried.

To a stirred solution of 500 mg. dicyclohexylcarbodiimide and 1 g. benzyl G-aminopenicillanate in 2 ml. methylene chloride is added 374 mg. p-guanidinomethylbenzoic acid hydrochloride in 2 ml. dimethylformamide. -The resulting mixture is stirred at 25 C. for approximately 30 minutes and subsequently at 0 to 5 C. for approximately 2 hours. Methylene chloride is added to the reaction mixture to precipitate dicyclohexylurea, which is removed by filtration. To the filtrate containing the product is added ethyl ether to precipitate p-guanidinomethylphenyl penicillin benzyl ester hydrochloride. The precipitated product is isolated in substantially pure form by further trituration with additional amounts of ether.

EXAMPLE 5 p-Guanidin omethylbenzyl penicillin Following the procedures described in the preceding example, p-cyanophenylacetic acid is converted by catalytic hydrogenation to p-aminomethylphenylacetic acid which is, in turn, reacted with o-methylisourea hydrochloride in concentrated ammonium hydroxide to produce p-guanidinomethylphenylacetic acid which is isolated as the hydrochloride. This product is contacted with benzyl 6- aminopenicillanate in the presence of dicyclohexylcarbodiimide to form the benzyl ester of p-guanidinomethylbenzyl penicillin hydrochloride which is hydrogenated in the presence of palladium catalyst to yield p-guanidinomethylbenzyl penicillin.

9 EXAMPLE 6 p-Guanidinolrnzyl penicillin A mixture of 0.2 g. of p-guanidinophenylacetic acid I and 0.3 ml. of thionyl chloride is prepared and allowed to material is separated from the supernatant liquid, washed with ether and dried, leaving substantially pure p-guanidinophenylacetyl chloride hydrochloride. M.P. 127-130 C. dec.

To the product obtained in this manner is added, with stirring, 0.23 g. of sodium G-amin-openicillanate in a mixture of 1 ml. of tetrahydrofuran and 1 ml. of water, followed by 2 ml. of 0.5 N aqueous sodium bircarbonate solution. The mixture is stirred for minutes at a temperature of about 25 C. and centrifuged, the product remaining in the supernatant liquid. The supernatant liquid is evaporated to dryness in vacuo, leaving as a residue p-guanidinobenzyl penicillin, which 'is further purified by washing with sodium bicarbonate solution and water, and dried. M.P. l95-200 dec. The product thus obtained inhibits Staphylococcus aureus at a concentration of 0.24 meg/ml.

The p-guanidinophenylacetic acid employed in the above reaction is prepared from p-aminophenylacetic acid by reaction with 1-nitro-2-methylisothiourea to yield as an intermediate product p-nitroguanidinophenylacetic acid, which is catalytically reduced in the presence of Raney nickel to yield as a product p-guanidinophenylacetic acid in accordance with the following procedure.

To a solution of 6.04 g. of p-aminophenylacetic acid in 50 ml. of water and ml. of 2.7 N sodium hydroxide is added5.4 g. of finely-ground 1-nitro-2-methylisothiourea. The mixture is stirred at room temperature for 16 hours, filtered, then carefully acidified with cooling. The product which crystallizes out is recrystallized from water, giving 4.4 g. of p-nitroguanidirrophenylacetic acid. M.P. 188-190".

One gram of Raney nickel is added to a hot solution of 2.0 g. of p nitroguanidinophenylacetie acid in 75 ml. of methanol and the mixture is refluxed for 15 minutes. The combined filtered solution and washings (79 ml.) is mixed with 1 gram fresh Raney nickel and hydrogenated at approximately 40 pounds per square inch pressure at 25 C.

Hydrogen absorption is complete after three hours. The

hydrogenation reaction mixture is filtered to remove the catalyst. The filtrate, containing p-guanidinophenylacetic acid, is evaporated to dryness in vacuo, and the residue crystallized from water. The first crop weighs 550 mg., gives a positive Sakaguchi test, and melts with decomposition at 320-2" C.

EXAMPLE 7 2,6-dimethoxy-4-guanidin0phenyl penicillin A solution of 274 mg. of 2,6-dirnethoxy-4-guanidinobenzoic acid hydrochloride dissolved in 1 ml. of dimethyl formamide is added to a solution containing0.31 g. 6- aminopencillanic acid benzyl ester and 0.25 g. dicyclohexylcarbodiimide dissolved in 0.5 ml. of dimethyl formamide. The resulting reaction mixture is maintained at 25 C. for /2 hour and filtered to remove dicyclohexylurea obtained as a by-product. The filtered dicyclohexylurea containing occluded product is washed with approximately 2 ml. of methylene chloride to dissolve additional product and the washings and filtrate combined. To the filtrate is added 100 ml. of ether and the material which separates from solution triturated with ether to solidify the product, benzyl 6 (2,6 dimethoxy 4 guanidinobenzamido)-penicillanate hydrochloride. The solid product thus obtained is recovered by filtration and washed with ether.

A solution of 300 mg. of the product resulting from the preceding reaction in 15 ml. of by volume aqueous methanol is hydrogenated under 40 p.s.i.g. hydrogen in the presence of 10% palladium-on-charcoal catalyst. for approximately 2 hours. The catalyst is removed by filtration and the filtrate containing the product neutralized to pH 7 with aqueous sodium bicarbonate solution. The filtrate containing the product is then evaporated to dryness in vacuo to obtain the residual solid comprising 2,6-dimethoxy-4-guanidinophenyl penicillin.

The 2,6-dimethoxy-4-guanidinobenzoic acid hydrochloride used in the preparation of the penicillins is prepared from 3-bromo-2,6-dimethoxybenzoic acid by first converting the acid to its benzyl ester, followed by reaction of the resulting benzyl ester with sodium amide in liquid ammonia to produce as an intermediate product benzyl-4-amino-2,6-dimethoxybenzoate which is, in turn, reacted with benzoyl cyanamide followed by hydrolysis, and the hydrolyzed product recovered by treatment with hydrochloric acid as 2,6-dimethoxy-4-guanidinobenzoic acid hydrochloride, in accordance with the following procedures.

A mixture of 11 g. of 3-bromo-2,6-dimethoxybenzoic acid and 3.7 g. of potassium carbonate and 6 ml. of benzyl bromide in ml. of acetone is heated at the reflux temperature of the mixture for approximately 20 hours. The reaction mixture is filtered and the filtrate containing the product benzyl-3-bromo-2,6-dimethoxybenzoate evaporated to remove the acetone solvent, leaving the product as an oily residue. The product is purified by dissolving in 100 ml. ether, washing with aqueous sodium bicarbonate, and removal of the ether and unreacted benzyl bromide by concentration under reduced pressure (100 C. at 0.1 mm.) for approximately one hour to obtain the product as a residue.

To a solution of sodium amide prepared by the reaction of 1.2 g. sodium and 200 ml. liquid ammonia is added, with stirring, 11 g. of benzyl-3-bromo-2,6-dimethoxybenzoate, and stirring is continued for approximately 3 /2 hours. To the reaction mixture is added 3.5 g. of ammonium chloride, and the ammonia removed by evaporation. The residue containing the product benzyl-4- amino-2,6-dimethoxybenzoate is extracted with a mixture of ml. ether and 150 ml. water, and the undissolved product recovered by filtration. Additional product is ob-' tained from the ether extract by back extraction with aqueous dilute sulfuric acid. The aqueous acid extract is then neutralized to precipitate additional product. The combined product is then recrystallized from methanol. M.P. 1l5116 C. The N.M.R. spectrum confirms that the thus-obtained product is benzyl-4-amino-2,6-dimethoxybenzoate.

A mixture of 574 mg. of benzyl-4-amino-2,6-dirnethoxybenzoate and 324 mg. of benzoyl cyanamide in 5 ml. of ethanol is heated at steam bath temperature for 30 minutes, during which time the ethanol evaporates. The residue is diluted with an additional 5 ml. of ethanol and evaporation and heating are continued for an additional 1% hours. The residual material is then contacted with 5 ml. ethanol and 0.5 ml. of 11.7 N sodium hydroxide solution and the resulting mixture heated for approximately 45 minutes and then allowed to stand at 25 C. for approximately 18 hours. The residue thus obtained is dissolved in 20 ml. water and the water solution of the product washed with 20 ml. of ether.' The aqueous solution of the product is acidified to about pH 2 and washed with ether to remove benzoic acid. The aqueous solution is then concentrated to 10 ml. and neutralized with aqueous sodium bicarbonate to precipitate 2,6-dimethoxy-4-guanidinobenzoic acid which is recovered by filtration and washed with water. The product thus obtained melts with decomposition at about 220 C.

The product thus obtained is dissolved in 1 ml. of aqueous hydrochloric acid and the solution evaporated to dryness to obtain as a residue 2,6-dimethoxy-4-guanidinobenzoic acid hydrochloride which is crystallized by stirring with acetone and recovered by filtration and washed With ether. M.P. dec. 220 C.

Various changes and modifications of the invention can be made, and to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope of the appended claim.

We claim: 5 methyl 3 (p guanidinophenyl) 4 isoxazolylcarboxylic acid.

No references cited.

HENRY R. JILES, Acting Primary Examiner.

R. J. GALLAGHER, Assistant Examiner. 

